RNA interference (RNAi) is an evolutionarily conserved cellular mechanism of post-transcriptional gene silencing found in fungi, plants and animals that uses small RNA molecules to inhibit gene expression in a sequence-specific manner RNAi is controlled by the RNA-induced silencing complex (RISC) that is initiated by short double-stranded RNA molecules in a cell's cytoplasm. The short double-stranded RNA interacts with Argonaute 2 (Ago2), the catalytic component of RISC, which cleaves target mRNA that is complementary to the bound RNA. One of the two RNA strands, known as the guide strand, binds the Ago2 protein and directs gene silencing, while the other strand, known as the passenger strand, is degraded during RISC activation. See, for example, Zamore and Haley, 2005, Science, 309:1519-1524; Vaughn and Martienssen, 2005, Science, 309:1525-1526; Zamore et al., 2000, Cell, 101:25-33; Bass, 2001, Nature, 411:428-429; and, Elbashir et al., 2001, Nature, 411:494-498. Single-stranded short interfering RNA has also been shown to bind Ago2 and support cleavage activity (see, e.g., Lima et al., 2012, Cell 150:883-894).
The RNAi machinery can be harnessed to destroy any mRNA of a known sequence. This allows for suppression (knockdown) of any gene from which it was generated, consequently preventing the synthesis of the target protein. Modulation of gene expression through an RNAi mechanism can be used to modulate therapeutically relevant biochemical pathways, including ones which are not accessible through traditional small molecule control.
Chemical modification of nucleotides incorporated into RNAi molecules leads to improved physical and biological properties, such as nuclease stability (see, e.g., Damha et al., 2008, Drug Discovery Today, 13:842-855), reduced immune stimulation (see, e.g., Sioud, 2006, TRENDS in Molecular Medicine, 12:167-176), enhanced binding (see, e.g., Koller, E. et al., 2006, Nucleic Acid Research, 34:4467-4476), and enhanced lipophilic character to improve cellular uptake and delivery to the cytoplasm. Thus, chemical modifications have the potential to increase potency of RNA compounds, allowing lower doses of administration, reducing the potential for toxicity, and decreasing overall cost of therapy.
In recent years, advances in oligonucleotide design and chemical modification types/patterns have resulted in molecules with increased resistance to nuclease-mediated degradation, improved pharmokinetics, increased gene specificity and reduced immunostimulatory responses (Lares, M. R. et al. 2010, Trends Biotechnol. 58:570-9). Despite these major advances, siRNA delivery to a diverse range of tissues remains a major obstacle in vivo. While siRNA delivery in vivo has been achieved in eye, lung, brain, tumor, and muscle by localized delivery (by intraocular, intranasal, intrathecal, intratumoral, and intramuscular injections, respectively), this delivery method is only suitable for target validation studies due to its invasive nature and has limited relevance as a clinical therapy (Golzio, M. et al., 2005, Gene Ther. 12:246-51; Liang, Y. et al., 2010, PLoS One 5:e12860; Reich, S. J. et al., 2003, Mol. Vis. 9:210-6; Tan, P. H. et al., 2005, Gene Ther. 12:59-66; Zhang, X. et al., 2004, J. Biol. Chem. 279:10677-84). A good systemic delivery system is essential to reach certain tissues of interest. Numerous studies have demonstrated systemic and targeted systemic siRNA delivery in vivo through a variety of methods, including cationic lipid and polymers, cholesterol conjugates, cell-penetrating peptides, recombinant viral vectors, small molecule carriers, antibody-linked siRNA and targeting ligands (Frank-Kamenetsky, M. et al., 2008, Proc. Natl. Acad. Sci. USA 105:11915-20; Khoury, M. et al., 2006, Arthritis Rheum. 54:1867-77; Kim, B. et al., 2004, Am. J. Pathol. 165:2177-85; Kondo, E. et al., 2012, Nat. Commun. 3:951; Morrissey, D. V. et al., 2005, Nat. Biotechnol. 23:1002-7; Schiffelers, R. M. et al., 2004, Nucleic Acids Res. 32:e149; Song, E. et al., 2005, Nat. Biotechnol. 23:709-17; Wolfrum, C. S. et al., 2007, Nat. Biotechnol. 25:1149-57). However, systemic siRNA delivery has remained limited to particular tissues, such as liver, tumors, spleen and jejunum (Abrams, M. T. et al., 2010, Mol. Ther. 18:171-80; Chien, P. Y. et al., 2005, Cancer Gene Ther. 12:321-8; Liang, Y. et al., supra; Sorensen, D. R. et al., 2003, J. Mol. Biol. 327:761-6; Tadin-Strapps, M. et al., 2011, J. Lipid Res. 52:1084-97; Wolfrum, C. et al., supra).
Myostatin is an inhibitor of skeletal muscle differentiation and growth. During development it is an inhibitor of myogenesis, while during adulthood its major role is in negatively regulating satellite cell activation and self-renewal. Myostatin is a member of the TGF-β family and acts as a catabolic stimulus through the ActRIIB receptor to induce SMAD2/3/FOXO/NF-κB signaling and muscle fiber atrophy (Sartori, R. G. et al., 2009, Am. J. Physiol. Cell Physiol. 296:C1248-57; Stitt, T. N. et al., 2004, Mol. Cell 14:395-403). Myostatin knockout mice, as well as other mouse models of myostatin inhibition, display increased muscle mass/strength and an attenuated/reversal of a muscle atrophy phenotype in different muscle disease models (Akpan, I. et al., 2009, Int. J. Obes. (Lond) 33:1265-73; Heineke, J. et al., 2010, Circulation 121:419-25; Lin, J. et al., 2002, Biochem. Biophys. Res. Commun. 291:701-6; Zhang, L. 2011, Faseb J. 25:1653-63; Zhou, X. et al., 2010, Cell 142:531-43). Small-interfering RNAs targeting myostatin may have numerous therapeutic applications in the multitude of existing muscle disorders, which range from muscular dystrophy, muscular atrophy in cachexia-inducing diseases, such as cancer, heart disease, chronic obstructive pulmonary disease, sarcopenia, chronic kidney disease, and metabolic diseases, and also in insulin-resistant disorders (Asp, M. L. et al., 2010, Int. J. Cancer 126:756-63; Bailey, J. L. et al., 2006, J. Am. Soc. Nephrol. 17:1388-94; Engelen, M. P. et al., 1994, Eur. Respir. J. 7:1793-7; Ruegg, M. A. et al., 2011, Annu. Rev. Pharmacol. Toxicol. 51:373-95).
To date there has been limited success in siRNA or antisense oligonucleotide (ASO) delivery systemically to muscle, with most reports highlighting muscle targeting by local injection (Gebski, B. L. et al., 2003, Hum. Mol. Genet. 12:1801-11; Guess, M. G. et al., 2013, Skelet. Muscle 3:19; Laws, N. et al., 2008, J. Appl. Physiol. 105:662-8; Tang, Y. et al., 2012, Mol. Pharmacol. 82:322-32). Several studies have used electroporation additively with intramuscular (IM) injections to improve the transfer of siRNAs or plasmid vectors into muscle cells (Eefting, D. et al., 2007, Hum. Gene Ther. 18:861-9; Golzio, M. et al., 2005, supra; Kishida, T. et al., 2004, J. Gene Med. 6:105-10). However, IM injections have a long-standing history for causing pain, local muscle damage and inflammation, which also minimizes their usefulness for therapeutic applications (McMahon, J. M. et al., 1998, Gene Ther. 5:1283-90). As an improvement to IM delivery, a model of “local” venous delivery muscle system was developed, which involves the use of a tourniquet to transiently isolate the injection solution in the muscle of the limb, in order to deliver a “high pressure” hydrodynamic injection of a luciferase pDNA vector to muscle in rats, dogs and monkeys (Hagstrom, J. E. et al., 2004, Mol. Ther. 10:386-98). Although it showed successful delivery into multiple muscle groups in the limb and the ability for multiple dosing, delivery efficiency was low and it is still an invasive technique that requires a high degree of injection skill.
In recent years, the use of the carrier polymer, atelocollagen, has been used for delivery of nucleic acids (siRNA, ASOs and plasmids) and negatively-charged proteins. Recent studies shows both local and systemic delivery of an atelocollagen/siRNA complex to muscle in a model of Duchenne muscular dystrophy (DMD) (Kawakami, E. et al., 2013, PLoS One 8:e64719; Kawakami, E. et al., 2011, Dev. Growth Differ. 53:48-54; Kinouchi, N. et al., 2008, Gene Ther. 15:1126-30).
There continues to be a need to develop therapies that can easily and non-invasively deliver nucleic acids to the muscle, which could have the potential for use in the future treatment of a variety of muscle disorders, such as muscular atrophic diseases, muscular dystrophy, and type II diabetes.